Influence of Temperature on Susceptibility of CVS. Tifguard and Georgia-06G Peanut to Meloidogyne arenaria.

Tifguard was released in 2008 as a peanut cultivar with a high level of resistance to Meloidogyne arenaria. Our objective was to determine the role of temperature on infection and development of M. arenaria in Tifguard compared to that in the nematode susceptible cultivar, Georgia-06G. Temperature affected the rate of nematode infection and development in both Tifguard and Georgia-06G (P ≤ 0.05). In Georgia-06G, egg-laying females were observed 25, 20 or 25 days after inoculation at 28°C, 31°C, and 34°C, respectively. There were greater numbers of nematodes entering roots and acceleration of development in response to 31°C compared with that at 28°C. There was, however, a decrease in the number of nematodes entering roots and their development was retarded at 34°C compared with that occurring at 31°C. Although second-stage juveniles penetrated Tifguard roots, they did not develop further at 28°C or 31°C; however, at 34°C both females, males, and a few egg-laying females of M. arenaria were observed. The optimum temperature for nematode infection and development was 31°C in Georgia-06G. In summary, it is unlikely that high soil temperatures would lessen the effectiveness of the nematode resistance gene in Tifguard.

Mapping Late Leaf Spot Resistance in Peanut (Arachis hypogaea) Using QTL-seq Reveals Markers for Marker-Assisted Selection.

Late leaf spot (LLS; Cercosporidium personatum) is a major fungal disease of cultivated peanut (Arachis hypogaea). A recombinant inbred line population segregating for quantitative field resistance was used to identify quantitative trait loci (QTL) using QTL-seq. High rates of false positive SNP calls using established methods in this allotetraploid crop obscured significant QTLs. To resolve this problem, robust parental SNPs were first identified using polyploid-specific SNP identification pipelines, leading to discovery of significant QTLs for LLS resistance. These QTLs were confirmed over 4 years of field data. Selection with markers linked to these QTLs resulted in a significant increase in resistance, showing that these markers can be immediately applied in breeding programs. This study demonstrates that QTL-seq can be used to rapidly identify QTLs controlling highly quantitative traits in polyploid crops with complex genomes. Markers identified can then be deployed in breeding programs, increasing the efficiency of selection using molecular tools. Key Message: Field resistance to late leaf spot is a quantitative trait controlled by many QTLs. Using polyploid-specific methods, QTL-seq is faster and more cost effective than QTL mapping.

A Technique to Study Meloidogyne arenaria Resistance in Agrobacterium rhizogenes-Transformed Peanut.

A reliable peanut root transformation system would be useful to study the functions of genes involved in root biology and disease resistance. The objective of this study was to establish an effective protocol to produce composite plants mediated by Agrobacterium rhizogenes transformation. In total, 75% of transformed peanut seedlings produced an average of 2.83 transgenic roots per plant. Peanut seed had the highest germination rate after treatment in a chlorine gas chamber for 8 h compared with 16 h in chlorine gas or Clorox and mercuric chloride immersion treatments. High transformation efficiency was achieved when the wound site for A. rhizogenes inoculation was covered with vermiculite instead of enclosing the whole plant in a high humidity chamber. On average, 2.5 galls from Meloidogyne arenaria infection were formed per transgenic root from susceptible genotype TifGP-2. These data indicate that A. rhizogenes-transformed roots can be used to phenotype the host response to nematode challenge. Transformation of RLP-2, a candidate resistance gene for M. arenaria integrated into a silencing construct, did not alter the resistance response of Tifguard, even though downregulation of endogenous RLP-2 expression was detected in transformed roots. It is likely that RLP-2 is not the gene conditioning M. arenaria resistance in peanut.

Response of New Field-Resistant Peanut Cultivars to Twin-Row Pattern or In-Furrow Applications of Phorate for Management of Spotted Wilt.

Field experiments were conducted at Marianna, FL in 2006 and Tifton, GA in 2006 and 2007 to compare new peanut (Arachis hypogaea) cultivars to the moderately resistant cv. Georgia Green and the highly resistant cv. AP-3 for field resistance to Tomato spotted wilt virus (TSWV), genus Tospovirus, and to determine the effects of in-furrow application of phorate insecticide and use of twin-row versus single-row patterns on incidence of spotted wilt in these cultivars. Cvs. Georgia Green, AP-3, Georgia-03L, Georgia-01R, Florida-07, McCloud, and York were evaluated in all five experiments, and Tifguard was added in experiments at Tifton. All cultivars except McCloud had lower incidence of spotted wilt than Georgia Green in all experiments. McCloud was intermediate in resistance to TSWV and had lower incidence of spotted wilt than Georgia Green in four of five experiments. Use of the twin-row pattern also reduced incidence of spotted wilt in McCloud in both years. On Georgia Green, phorate reduced incidence of spotted wilt in 2007 and twin-row pattern reduced incidence in both years. Phorate had no effect on spotted wilt in AP-3, Georgia-03L, McCloud, Georgia-01R, or Tifguard in either year. Twin-row pattern reduced either final incidence or area under the disease progress curve in all cultivars in at least 1 year of the study. All of these new cultivars should reduce the risk of losses to spotted wilt compared with Georgia Green. In highly resistant cultivars, especially AP-3, York, and Tifguard, use of phorate insecticide or twin-row pattern may not be necessary, and may not provide noticeable benefit in reduction of spotted wilt or increased yield.

Resistance in Peanut Cultivars and Breeding Lines to Three Root-Knot Nematode Species.

Three major species of root-knot nematode infect peanut: Meloidogyne arenaria race 1, M. hapla, and M. javanica race 3. Sources of resistance to all three nematodes are needed for developing novel peanut cultivars with broad resistance to Meloidogyne spp. Cultivars and breeding lines of peanut were evaluated for resistance to M. arenaria, M. hapla, and M. javanica in the greenhouse and in the laboratory. Twenty-six genotypes with some resistance to M. arenaria, M. javanica, or M. hapla were identified from 60 accessions based on average eggs per gram of root and gall index relative to a susceptible control. Among these, 14 genotypes were moderately to highly resistant to all three species, 5 genotypes were resistant to M. arenaria and M. javanica, 2 genotypes were resistant to M. javanica and M. hapla, 1 genotype was resistant M. arenaria alone, and 4 genotypes were resistant to M. hapla alone. Reproduction of M. arenaria on lines NR 0817, C724-19-11, and D108 was highly variable, indicating that these genotypes likely were heterogeneous for resistance. COAN, NemaTAM, C724-25-8, and the M. arenaria-resistant plants of C724-19-11 contained the dominant sequence-characterized amplified region marker (197/909) for nematode resistance. Results with the molecular markers indicate that the high resistance to M. arenaria in GP-NC WS 6 may be different from the resistance in COAN, NemaTAM, and C724-25-8. Resistance to M. arenaria was correlated with resistance to M. javanica in peanut, whereas resistance to M. hapla was not correlated with the resistance to either M. arenaria or M. javanica. The resistant selections should be valuable sources for pyramiding resistance genes to develop new cultivars with broad and durable resistance to Meloidogyne spp.

Storage protein profiles in Spanish and runner market type peanuts and potential markers.

BACKGROUND: Proteomic analysis has proven to be the most powerful method for describing plant species and lines, and for identification of proteins in complex mixtures. The strength of this method resides in high resolving power of two-dimensional electrophoresis (2-DE), coupled with highly sensitive mass spectrometry (MS), and sequence homology search. By using this method, we might find polymorphic markers to differentiate peanut subspecies. RESULTS: Total proteins extracted from seeds of 12 different genotypes of cultivated peanut (Arachis hypogaea L.), comprised of runner market (A. hypogaea ssp. hypogaea) and Spanish-bunch market type (A. hypogaea ssp. fastigiata), were separated by electrophoresis on both one- and two-dimensional SDS-PAGE gels. The protein profiles were similar on one-dimensional gels for all tested peanut genotypes. However, peanut genotype A13 lacked one major band with a molecular weight of about 35 kDa. There was one minor band with a molecular weight of 27 kDa that was present in all runner peanut genotypes and the Spanish-derivatives (GT-YY7, GT-YY20, and GT-YY79). The Spanish-derivatives have a runner-type peanut in their pedigrees. The 35 kDa protein in A13 and the 27 kDa protein in runner-type peanut genotypes were confirmed on the 2-D SDS-PAGE gels. Among more than 150 main protein spots on the 2-D gels, four protein spots that were individually marked as spots 1-4 showed polymorphic patterns between runner-type and Spanish-bunch peanuts. Spot 1 (ca. 22.5 kDa, pI 3.9) and spot 2 (ca. 23.5 kDa, pI 5.7) were observed in all Spanish-bunch genotypes, but were not found in runner types. In contrast, spot 3 (ca. 23 kDa, pI 6.6) and spot 4 (ca. 22 kDa, pI 6.8) were present in all runner peanut genotypes but not in Spanish-bunch genotypes. These four protein spots were sequenced. Based on the internal and N-terminal amino acid sequences, these proteins are isoforms (iso-Ara h3) of each other, are iso-allergens and may be modified by post-translational cleavage. CONCLUSION: These results suggest that there may be an association between these polymorphic storage protein isoforms and peanut subspecies fastigiata (Spanish type) and hypogaea (runner type). The polymorphic protein peptides distinguished by 2-D PAGE could be used as markers for identification of runner and Spanish peanuts.

Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea).

Preharvest aflatoxin contamination has been identified by the peanut industry as a serious issue in food safety and human health because of the carcinogenic toxicity. Drought stress is the most important environmental factor exacerbating Aspergillus infection and aflatoxin contamination in peanut. The development of drought-tolerant peanut cultivars could reduce aflatoxin contamination and would represent a major advance in the peanut industry. In this study, we identified a novel PLD gene in peanut (Arachis hypogaea), encoding a putative phospholipase D (PLD, EC 3.1.4.4). The completed cDNA sequence was obtained by using the consensus-degenerated hybrid oligonucleotide primer strategy. The deduced amino acid sequence shows high identity with known PLDs, and has similar conserved domains. The PLD gene expression under drought stress has been studied using four peanut lines: Tifton 8 and A13 (both drought tolerant) and Georgia Green (moderate) and PI 196754 (drought sensitive). Northern analysis showed that PLD gene expression was induced faster by drought stress in the drought-sensitive lines than the drought tolerance lines. Southern analysis showed that cultivated peanut has multiple copies (3 to 5 copies) of the PLD gene. These results suggest that peanut PLD may be involved in drought sensitivity and tolerance responses. Peanut PLD gene expression may be useful as a tool in germplasm screening for drought tolerance.

Identification of Transcripts Involved in Resistance Responses to Leaf Spot Disease Caused by Cercosporidium personatum in Peanut (Arachis hypogaea).

ABSTRACT Late leaf spot disease caused by Cercosporidium personatum is one of the most destructive foliar diseases of peanut (Arachis hypogaea) worldwide. The objective of this research was to identify resistance genes in response to leaf spot disease using microarray and real-time polymerase chain reaction (PCR). To identify transcripts involved in disease resistance, we studied the gene expression profiles in two peanut genotypes, resistant or susceptible to leaf spot disease, using cDNA microarray containing 384 unigenes selected from two expressed sequenced tag (EST) cDNA libraries challenged by abiotic and biotic stresses. A total of 112 spots representing 56 genes in several functional categories were detected as up-regulated genes (log(2) ratio > 1). Seventeen of the top 20 genes, each matching gene with known function in GenBank, were selected for validation of their expression levels using real-time PCR. The two peanut genotypes were also used to study the functional analysis of these genes and the possible link of these genes to the disease resistance trait. Microarray technology and real-time PCR were used for comparison of gene expression. The selected genes identified by microarray analysis were validated by real-time PCR. These genes were more greatly expressed in the resistant genotype as a result of response to the challenge of C. personatum than in the susceptible genotype. Further investigations are needed to characterize each of these genes in disease resistance. Gene probes could then be developed for application in breeding programs for marker-assisted selection.

beta-1,3-Glucanase Activity in Peanut Seed (Arachis hypogaea) is Induced by Inoculation with Aspergillus flavus and Copurifies with a Conglutin-Like Protein.

ABSTRACT Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein beta-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of beta-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of beta-1,3-glucanase revealed that there were more protein bands corresponding to beta-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic beta-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of beta-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having beta-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has beta-1,3-glucanase activity.

Relationship between Meloidogyne arenaria and Aflatoxin Contamination in Peanut.

Damaged and developing kernels of peanut (Arachis hypogaea) are susceptible to colonization by fungi in the Aspergillus flavus group which, under certain conditions, produces aflatoxins prior to harvest. Our objective was to determine whether infection of peanut roots and pods by Meloidogyne arenaria increases aflatoxin contamination of the kernels when peanut is subjected to drought stress. The experiment was a completely randomized 2-x-2 factorial with 6 replicates/treatment. The treatment factors were nematodes (plus and minus M. arenaria) and fungus (plus and minus A. flavus inoculum). The experiment was conducted in 2001 and 2002 in microplots under an automatic rain-out shelter. In treatments where A. flavus inoculum was added, aflatoxin concentrations were high (> 1,000 ppb) and not affected by nematode infection; in treatments without added fungal inoculum, aflatoxin concentrations were greater (P

Reproduction of Meloidogyne spp. on Resistant Peanut Genotypes from Three Breeding Programs.

Three described species of root-knot nematode parasitize peanut (Arachis hypogaea): Meloidogyne arenaria race 1 (Ma), M. hapla (Mh), and M. javanica (Mj). Peanut cultivars with broad resistance to Meloidogyne spp. will be useful regardless of the species present in the field. The objective of this study was to determine whether peanut genotypes with resistance to M. arenaria originating from three different breeding programs were also resistant to M. hapla and M. javanica. The experiment used a factorial arrangement (completely randomized) with peanut genotype and nematode population as the factors. The five peanut genotypes were 'COAN' and AT 0812 (highly resistant to Ma), C209-6-13 (moderately resistant to Ma), and 'Southern Runner' and 'Georgia Green' (susceptible to Ma). The four nematode populations were two isolates of Ma (Gibbs and Gop) and one isolate each of Mh and Mj. On COAN or AT 0812, both Ma and Mj produced <10% of the eggs produced on Georgia Green. On the peanut genotype C209-6-13, Ma and Mj produced about 50% of the eggs produced on Georgia Green. None of the resistant genotypes exhibited a high level of resistance to Mh. The lack of resistance to Mh in any cultivars or advanced germplasm is a concern because the identity of a Meloidogyne sp. in a particular peanut field is generally not known. Breeding efforts should focus on moving genes for resistance to M. hapla into advanced peanut germplasm, and combining genes for resistance to the major Meloidogyne spp. in a single cultivar.

Differential Response of Selected Peanut (Arachis hypogaea) Genotypes to Mechanical Inoculation by Tomato spotted wilt virus.

Screening of peanut germ plasm for resistance to Tomato spotted wilt virus (TSWV) has been largely inefficient due to the lack of a screening technique based on mechanical transmission of the virus under controlled environmental conditions. We have studied the reaction of three peanut cultivars (Georgia Green, Georgia Runner, C-99R) and one breeding line (C11-2-39) using a highly efficient mechanical inoculation procedure. The disease response was studied at two temperature regimes, 25 to 30°C (low temperature) and 30 to 37°C (high temperature). Based on percent transmission, symptomatology, distribution of TSWV, and relative levels of TSWV nucleocapsid (N) protein, Georgia Runner and Georgia Green were found to be susceptible, whereas C-99R and C11-2-39 were resistant. Of the four genotypes tested, C11-2-39 had the highest level of resistance to TSWV. The results correlated with the field performance of the genotypes except in the case of Georgia Green, which could not be distinguished from TSWV-susceptible Georgia Runner. Exposure of the inoculated plants to higher temperature (30 to 37°C) resulted in a better resistant response as reflected by reduced systemic infection, localized symptom expression, restricted viral movement, and reduced levels of TSWV antigen. To our knowledge, this is the first report of differential response of peanut genotypes to TSWV using mechanical inoculation. The four peanut genotypes should be useful as reference standards for the initial screening and identification of sources of TSWV resistance in peanut germ plasm.

Identification of Resistance to Rhizoctonia Limb Rot in a Core Collection of Peanut Germ Plasm.

Diseases caused by Rhizoctonia solani lead to significant reductions in peanut yields and quality throughout the world. A subset of accessions from the peanut germ plasm core collection plus the commercial cultivars Florunner, Southern Runner, Georgia Browne, and Georgia Green were evaluated for resistance to limb and seedling hypocotyl infections caused by R. solani. Georgia Green and core accessions 95 (PI 497351), 197 (PI 331326), 208 (PI 274193), 244 (PI 343361), 246 (PI 343398), and 524 (PI 288178) had levels of resistance comparable to Georgia Browne, the only commercial cultivar reported to have partial resistance to Rhizoctonia limb rot. Eleven core accessions, representing the full range of disease expression, and the commercial cultivars were evaluated in growth chambers to quantify their susceptibility to seedling hypocotyl infections and to determine if evaluating seedlings could serve as a primary screening method to identify potential sources of limb rot resistance. The most resistant core accessions to seedling hypocotyl infections were 234 (PI 159664) and 366 (PI 268968), and the most resistant commercial cultivar was Georgia Green. There was not a significant correlation between resistance to limb rot in the field and the severity of hypocotyl infections in growth chambers, indicating that resistance to hypocotyl infections is not a good indicator of resistance to Rhizoctonia limb rot.

Development of greenhouse screening for resistance to Aspergillus parasiticus infection and preharvest aflatoxin contamination in peanut.

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F2 generation. The heritability estimates of crosses GFA-2 x NC-V11 and Tifton-8 X NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.

Field Evaluation of Susceptibility to Meloidogyne arenaria in Arachis hypogaea Plant Introductions.

Resistance to Meloidogyne arenaria race 1 is not currently available in commercial peanut cultivars. Moderate levels of resistance have been identified in Arachis hypogaea plant introductions (PI) in previous greenhouse studies. The purpose of this work was to evaluate the effects of resistance in peanut PI on populations dynamics of M. arenaria in field plots. The PI designated as resistant in greenhouse studies had fewer M. arenaria in roots than the most susceptible PI. At midseason and at the end of the season, resistant PI had fewer M. arenaria in rhizosphere soil than the most susceptible PI. Seven resistant PI had lower numbers of M. arenaria than 'Florunner' at the end of the growing season. Gall index, egg mass index, number of eggs/plant, and number of eggs/g root from greenhouse screening were highly correlated with population levels of M. arenaria in the field, especially at midseason. These greenhouse indices should provide reliable estimates of host suitability in future studies.